TNFα-neutralizing antibodies

ABSTRACT

The invention provides monoclonal antibodies that neutralize TNFα activity. The monoclonal antibodies may be rabbit monoclonal antibodies or monoclonal antibodies having CDR regions derived from those rabbit monoclonal antibodies. In certain embodiments, the monoclonal antibodies may be humanized. Methods of using the subject antibodies to inhibit TNFα activity, methods of treatment using those antibodies and kits containing the same are also provided. The invention finds use in a variety of research and medical applications.

CROSS-REFERENCING

This application is a continuation of application Ser. No. 12/196,200,filed Aug. 21, 2008, which application is a continuation of applicationSer. No. 11/090,105, filed Mar. 24, 2005, and now issued as U.S. Pat.No. 7,431,927.

INTRODUCTION

1. Field of the Invention

The field of this invention is antibodies, particularly monoclonalantibodies that neutralize tumor necrosis factor-α (TNFα) activity.

2. Background of the Invention

The pathology of a variety of disorders is attributed to excessiveamounts of TNF-α, either locally or systemically. For example, there isstrong evidence that abnormally high production and release from cellsof TNF-α contributes to disease initiation and progression in rheumatoidarthritis, systemic inflammatory syndromes, diabetes, and multiplesclerosis. In every one of these conditions, the initiating andsustaining pathophysiologic action is directly a result of an immediatelocal release and synthesis of massive amounts of TNF-α from severaltypes of cells at or adjacent to the site of tissue damage. The locallyreleased TNF-α is followed by additional synthesis and release of TNF-αby invading macrophages drawn to the site of tissue damage by a cascadeof chemotactic cytokines released locally from cells in response to thegreatly elevated TNF-α concentrations.

There is a need in the art for methods of treating TNF-α-mediateddisorders. The present invention addresses this need.

LITERATURE

Literature of interest includes: published U.S. patent applications20040151722, 20050037008, 20040185047, 20040138427, 20030187231,20040002589 and 20030199679 and Balazovich (Blood 1996 88: 690-696).

SUMMARY OF THE INVENTION

The invention provides monoclonal antibodies that neutralize TNFαactivity. The monoclonal antibodies may be rabbit monoclonal antibodiesor monoclonal antibodies having CDR regions derived from those rabbitmonoclonal antibodies. In certain embodiments, the monoclonal antibodiesmay be humanized. Methods of using the subject antibodies to inhibitTNFα activity, methods of treatment using those antibodies and kitscontaining the same are also provided. The invention finds use in avariety of research and medical applications.

These and other advantages and features of the invention will becomeapparent to those persons skilled in the art upon reading the details ofthe invention as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. shows a sequence alignment of the heavy chain amino acidsequences of 44 exemplary rabbit TNFα neutralizing antibodies. Variousantibody domains are indicated.

FIG. 1B. shows a sequence alignment of the light chain amino acidsequences of 44 exemplary rabbit TNFα neutralizing antibodies. The lightchains set forth in this figure are partnered with corresponding heavychains in FIG. 1A to provide antibodies that neutralizes TNFα. Variousantibody domains are indicated.

FIG. 2A shows the number of times an amino acid is present at eachposition of the heavy chains set forth in FIG. 1A. The amino acidpositions are numbered using standard Kabat numbering, supra. Variousantibody domains are indicated.

FIG. 2B shows the number of times an amino acid is present at eachposition of the light chains set forth in FIG. 1B. The amino acidpositions are numbered using standard Kabat numbering. Various antibodydomains are indicated.

FIG. 3A shows sequence alignments for heavy chains of each of 7 groupsof related antibodies (i.e., antibodies produced by cells having acommon naïve B cell ancestor).

FIG. 3B shows sequence alignments for heavy chains of each of 7 groupsof related antibodies (i.e., antibodies produced by cells having acommon naïve B cell ancestor).

DEFINITIONS

Before the present subject invention is described further, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anantibody” includes a plurality of such antibodies and reference to “aframework region” includes reference to one or more framework regionsand equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. These terms are well understood by those in the field, and referto a protein consisting of one or more polypeptides that specificallybinds an antigen. One form of antibody constitutes the basic structuralunit of an antibody. This form is a tetramer and consists of twoidentical pairs of antibody chains, each pair having one light and oneheavy chain. In each pair, the light and heavy chain variable regionsare together responsible for binding to an antigen, and the constantregions are responsible for the antibody effector functions.

The recognized immunoglobulin polypeptides include the kappa and lambdalight chains and the alpha, gamma (IgG₁, IgG₂, IgG₃, IgG₄), delta,epsilon and mu heavy chains or equivalents in other species. Full-lengthimmunoglobulin “light chains” (of about 25 kDa or about 214 amino acids)comprise a variable region of about 110 amino acids at the NH₂-terminusand a kappa or lambda constant region at the COOH-terminus. Full-lengthimmunoglobulin “heavy chains” (of about 50 kDa or about 446 aminoacids), similarly comprise a variable region (of about 116 amino acids)and one of the aforementioned heavy chain constant regions, e.g., gamma(of about 330 amino acids).

The terms “antibodies” and “immunoglobulin” include antibodies orimmunoglobulins of any isotype, fragments of antibodies which retainspecific binding to antigen, including, but not limited to, Fab, Fv,scFv, and Fd fragments, chimeric antibodies, humanized antibodies,single-chain antibodies, and fusion proteins comprising anantigen-binding portion of an antibody and a non-antibody protein. Theantibodies may be detectably labeled, e.g., with a radioisotope, anenzyme which generates a detectable product, a fluorescent protein, andthe like. The antibodies may be further conjugated to other moieties,such as members of specific binding pairs, e.g., biotin (member ofbiotin-avidin specific binding pair), and the like. The antibodies mayalso be bound to a solid support, including, but not limited to,polystyrene plates or beads, and the like. Also encompassed by the termare Fab′, Fv, F(ab′)₂, and or other antibody fragments that retainspecific binding to antigen, and monoclonal antibodies.

Antibodies may exist in a variety of other forms including, for example,Fv, Fab, and (Fab′)₂, as well as bi-functional (i.e. bi-specific) hybridantibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987))and in single chains (e.g., Huston et al., Proc. Natl. Acad. Sci.U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426(1988), which are incorporated herein by reference). (See, generally,Hood et al., “Immunology”, Benjamin, N.Y., 2nd ed. (1984), andHunkapiller and Hood, Nature, 323, 15-16 (1986)).

An immunoglobulin light or heavy chain variable region consists of a“framework” region (FR) interrupted by three hypervariable regions, alsocalled “complementarity determining regions” or “CDRs”. The extent ofthe framework region and CDRs have been precisely defined (see,“Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S.Department of Health and Human Services, (1991)). The numbering of allantibody amino acid sequences discussed herein conforms to the Kabatsystem. The sequences of the framework regions of different light orheavy chains are relatively conserved within a species. The frameworkregion of an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs. The CDRs are primarily responsible for binding to an epitope of anantigen.

Chimeric antibodies are antibodies whose light and heavy chain geneshave been constructed, typically by genetic engineering, from antibodyvariable and constant region genes belonging to different species. Forexample, the variable segments of the genes from a rabbit monoclonalantibody may be joined to human constant segments, such as gamma 1 andgamma 3. An example of a therapeutic chimeric antibody is a hybridprotein composed of the variable or antigen-binding domain from a rabbitantibody and the constant or effector domain from a human antibody(e.g., the anti-Tac chimeric antibody made by the cells of A.T.C.C.deposit Accession No. CRL 9688), although other mammalian species may beused.

As used herein, the term “humanized antibody” or “humanizedimmunoglobulin” refers to an non-human (e.g., mouse or rabbit) antibodycontaining one or more amino acids (in a framework region, a constantregion or a CDR, for example) that have been substituted with acorrespondingly positioned amino acid from a human antibody. In general,humanized antibodies produce a reduced immune response in a human host,as compared to a non-humanized version of the same antibody.

It is understood that the humanized antibodies designed and produced bythe present method may have additional conservative amino acidsubstitutions which have substantially no effect on antigen binding orother antibody functions. By conservative substitutions is intendedcombinations such as those from the following groups: gly, ala; val,ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Aminoacids that are not present in the same group are “substantiallydifferent” amino acids.

The term “specific binding” refers to the ability of an antibody topreferentially bind to a particular analyte that is present in ahomogeneous mixture of different analytes. In certain embodiments, aspecific binding interaction will discriminate between desirable andundesirable analytes in a sample, in some embodiments more than about 10to 100-fold or more (e.g., more than about 1000- or 10.000-fold).

In certain embodiments, the affinity between a capture agent and analytewhen they are specifically bound in a capture agent/analyte complex ischaracterized by a K_(D) (dissociation constant) of less than 10⁻⁶M,less than 10⁻⁷ M, less than 10⁻⁸ M, less than 10⁻⁹ M, less than 10⁻⁹ M,less than 10⁻¹¹ M, or less than about 10⁻¹² M or less.

An amino acid residue that is in “close contact”, “close proximity” or“in close proximity to” another amino acid residue is an amino acidresidue that is has a side chain that is close to, i.e., within 7, 6, 5or 4 Angstroms of, a side chain of another amino acid. For example, anamino acid that are proximal to a CDR is a non-CDR amino acid that has aside chain that is close to a side chain of an amino acid in a CDR.

A “variable region” of a heavy or light antibody chain is an N-terminalmature domain of the chains. All domains, CDRs and residue numbers areassigned on the basis of sequence alignments and structural knowledge.Identification and numbering of framework and CDR residues is asdescribed in by Chothia and others (Chothia, Structural determinants inthe sequences of immunoglobulin variable domain. J Mol Biol 1998;278:457-79).

VH is the variable domain of an antibody heavy chain. VL is the variabledomain of an antibody light chain, which could be of the kappa (K) or ofthe lambda isotype. K-1 antibodies have the kappa-1 isotype whereas K-2antibodies have the kappa-2 isotype and VL is the variable lambda lightchain.

A “buried residue” is an amino acid residue whose side chain has lessthan 50% relative solvent accessibility, which is calculated as thepercentage of the solvent accessibility relative to that of the sameresidue, X, placed in an extended GGXGG peptide. Methods for calculatingsolvent accessibility are well known in the art (Connolly 1983 J. appl.Crystallogr, 16, 548-558).

As used herein, the terms “determining,” “measuring,” and “assessing,”and “assaying” are used interchangeably and include both quantitativeand qualitative determinations.

The terms “polypeptide” and “protein”, used interchangeably herein,refer to a polymeric form of amino acids of any length, which caninclude coded and non-coded amino acids, chemically or biochemicallymodified or derivatized amino acids, and polypeptides having modifiedpeptide backbones. The term includes fusion proteins, including, but notlimited to, fusion proteins with a heterologous amino acid sequence,fusions with heterologous and homologous leader sequences, with orwithout N-terminal methionine residues; immunologically tagged proteins;fusion proteins with detectable fusion partners, e.g., fusion proteinsincluding as a fusion partner a fluorescent protein, β-galactosidase,luciferase, etc.; and the like. Polypeptides may be of any size, and theterm “peptide” refers to polypeptides that are 8-50 residues (e.g., 8-20residues) in length.

As used herein the term “isolated,” when used in the context of anisolated antibody, refers to an antibody of interest that is at least60% free, at least 75% free, at least 90% free, at least 95% free, atleast 98% free, and even at least 99% free from other components withwhich the antibody is associated with prior to purification.

The terms “treatment” “treating” and the like are used herein to referto any treatment of any disease or condition in a mammal, e.g.particularly a human or a mouse, and includes: a) preventing a disease,condition, or symptom of a disease or condition from occurring in asubject which may be predisposed to the disease but has not yet beendiagnosed as having it; b) inhibiting a disease, condition, or symptomof a disease or condition, e.g., arresting its development and/ordelaying its onset or manifestation in the patient; and/or c) relievinga disease, condition, or symptom of a disease or condition, e.g.,causing regression of the condition or disease and/or its symptoms.

The terms “subject,” “host,” “patient,” and “individual” are usedinterchangeably herein to refer to any mammalian subject for whomdiagnosis or therapy is desired, particularly humans. Other subjects mayinclude cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses,and so on.

“Corresponding amino acids”, as will be exemplified below, are aminoacid residues that are at an identical position (i.e., they lie acrossfrom each other) when two or more amino acid sequences are aligned.Methods for aligning and numbering antibody sequences are set forth ingreat detail in Chothia, supra, Kabat supra, and others. As is known inthe art (see, e.g. Kabat 1991 Sequences of Proteins of ImmunologicalInterest, DHHS, Washington, D.C.), sometimes one, two or three gapsand/or insertions of up to one, two, three or four residues, or up toabout 15 residues (particularly in the L3 and H3CDRs) may be made to oneor both of the amino acids of an antibody in order to accomplish analignment.

A “natural” antibody is an antibody in which the heavy and lightimmunoglobulins of the antibody have been naturally selected by theimmune system of a multi-cellular organism, as opposed to unnaturallypaired antibodies made by e.g. phage display, or humanized antibodies.As such, the subject parental antibodies do not usually contain anyviral (e.g., bacteriophage M13)-derived sequences. Spleen, lymph nodesand bone marrow are examples of tissues that produce natural antibodies.

A “substitutable position”, as will be described in greater detailbelow, is a particular position of an antibody that may be substitutedby different amino acids without significantly decreasing the bindingactivity of the antibody. Methods for identifying substitutablepositions, and how they may be substituted, are described in muchgreater detail below. A substitutable positions may also be referred toas “variation tolerant position”.

A “parent” antibody, as will be described in greater detail below, is anantibody is the target of amino acid substitutions. In certainembodiments, amino acids may be “donated” by a “donor” antibody to theparent antibody to produce an altered antibody.

“Related antibodies”, as will be described in greater detail below, areantibodies that have a similar sequence and produced by cells that havea common B cell ancestor. Such a B cell ancestor contains a genomehaving a rearranged light chain VJC region and a rearranged heavy chainVDJC region, and produces an antibody that has not yet undergoneaffinity maturation. “Naïve” or “virgin” B cells present in spleentissue, are exemplary B cell common ancestors. Related antibodies bindto the same epitope of an antigen and are typically very similar insequence, particularly in their L3 and H3CDRs. Both the H3 and L3 CDRsof related antibodies have an identical length and a near identicalsequence (i.e., differ by 0, 1 or 2 residues). Related antibodies arerelated via a common antibody ancestor, the antibody produced in thenaïve B cell ancestor. The term “related antibodies” is not intended todescribe a group of antibodies that do not have a common antibodyancestor produced by a B-cell.

The term “TNFα” or its non-abbreviated form “tumor necrosis factor-α”,as used herein, is intended to refer to a human cytokine that exists asa 17 kD secreted form and a 26 kD membrane associated form, thebiologically active form of which is composed of a trimer ofnoncovalently bound 17 kD molecules. The structure of TNFα is describedfurther in, for example, Pennica et al. (Nature 1984 312:724-729), Daviset al. (Biochemistry 1987 26:1322-1326) and Jones et al. (Nature 1989338:225-228). The term TNFα is intended to include recombinant TNFαmolecules, which can be prepared by standard recombinant expressionmethods or purchased commercially (R & D Systems, Catalog No. 210-TA,Minneapolis, Minn.), as well as fusion proteins containing a TNFαmolecule. Amino acid sequences of exemplary TNFαs that may be employedherein are found in the NCBI's Genbank database and a full descriptionof human TNFα and its role in various diseases and conditions is foundin NCBI's Online Mendelian Inheritance in Man database.

A “TNFα neutralizing antibody”, “antibody that neutralizes TNFαactivity” or any grammatical equivalent thereof, is intended to refer toan antibody whose binding to TNFα results in inhibition of a biologicalactivity of TNFα. This inhibition of the biological activity of TNFα canbe assessed by measuring one or more indicators of TNFα biologicalactivity, such as TNFα-induced cytotoxicity (either in vitro or invivo), TNFα-induced cellular activation or TNFα binding to a TNFαreceptor. TNFα biological activity can be assessed by one or more ofseveral standard in vitro or in vivo assays known in the art.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The invention provides monoclonal antibodies that neutralize TNFαactivity. The monoclonal antibodies may be rabbit monoclonal antibodiesor monoclonal antibodies having CDR regions derived from those rabbitmonoclonal antibodies. In certain embodiments, the monoclonal antibodiesmay be humanized. Methods of using the subject antibodies to inhibitTNFα activity, methods of treatment using those antibodies and kitscontaining the same are also provided. The invention finds use in avariety of research and medical applications.

Rabbit Monoclonal Antibodies that Neutralize TNFα Activity

In one embodiment, the invention provides rabbit monoclonal antibodiesthat neutralize TNFα activity, where a rabbit monoclonal antibody is anatural full-length monoclonal rabbit antibody (i.e., an antibodyencoded by a rabbit and that that has been naturally selected by theimmune system of an immunized rabbit), or any antigen-binding portionthereof (i.e., is an antibody that contains at least all of theframework and complementary determining regions of the full-lengthantibody).

A TNFα neutralizing rabbit monoclonal antibody of the invention inhibitsat least one activity of TNFα in the range of about 20% to 100%, e.g.,by at least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, usually up to about70%, up to about 80%, up to about 90% or more. In any of these assays, asubject antibody inhibits TNFα activity with an IC₅₀ of 1×10⁻⁷ M or less(e.g., 1×10⁻⁷ M or less, 1×10⁻⁸ M or less, 1×10⁻⁹ M or less, usually to1×10⁻¹² M or 1×10⁻¹³ M). In assays in which a mouse is employed, asubject antibody typically has an ED₅₀ of less then 1 μg/mouse (e.g., 10ng/mouse to 1 μg/mouse).

TNFα activity can be assayed in a variety of ways, including, but notlimited to: assays for TNFα-induced cytotoxicity (either in vitro or invivo) using suitable cells, e.g., L929 cells; assays for binding of TNFαto its receptor using suitable cells, e.g., U-937 cells; assays forinhibition of endothelial cell leukocyte adhesion molecule 1 (ELAM-1)expression on human umbilical vein endothelian (HEVEC) cells; or in vivoassays using D-galactosamine sensitized mice. Such assays are describedin great detail in U.S. Pat. No. 6,090,382, which is incorporated byreference herein for that purpose.

The subject rabbit monoclonal antibodies have the following generalcharacteristics:

a) high affinity for TNFα (e.g., a K_(d) of 10⁻⁸ or less);

b) slow off rate for dissociation with TNFα (e.g., a K_(off) of 10⁻³sec⁻¹ or less); and

c) TNFα neutralizing activity.

Methods for measuring binding affinity, off rate and other antibodybinding kinetics are well known in the art, and may be employed todetermine whether an antibody has a high affinity and a slow off ratefor TNFα. In many methods and as is well known in the art, antibodybinding kinetics may be measured by ELISA methods or by measuringsurface plasmon resonance using, for example, a BIACORE™ biosensor soldby Pharmacia (now Pfizer). Methods for measuring binding of antigens toantibodies using surface plasmon resonance are well known in the art(see, e.g., Methods of Dev. Biol. 2003 112:141-51 and J. Mol. Recognit.1999 12:310-5) and are readily adapted for use herein.

In certain embodiments a subject rabbit monoclonal antibody has a heavychain that is substantially identical (e.g., at least about 70%, atleast about 80%, at least about 90%, at least about 95% or at leastabout 98% identical) to that of any of the heavy chain variable domainsequences set forth in FIG. 1A, and a light chain that is substantiallyidentical (e.g., at least about 70%, at least about 80%, at least about90%, at least about 95% or at least about 98% identical) to that of anyof the light chain variable domain sequences set forth in FIG. 1B. Inparticular embodiments, a subject rabbit monoclonal antibody hasframework sequences or CDRs that are substantially identical (e.g., atleast about 70%, at least about 80%, at least about 90%, at least about95% or at least about 98% identical) to the framework sequences or CDRsof any of the heavy or light chain sequences shown in FIG. 1A or 1B.

In certain embodiments, rabbit monoclonal antibodies of the inventionmay contain a heavy or light chain that is encoded by a polynucleotidethat hybridizes under high stringency conditions to a rabbit heavy orlight chain-encoding nucleic acid. High stringency conditions includeincubation at 50° C. or higher in 0.1×SSC (15 mM saline/0.15 mM sodiumcitrate).

In certain embodiments, rabbit monoclonal antibodies of the inventionmay contain a heavy or light chain that is encoded by a polynucleotidethat is at least 80% identical to (e.g., at least 85%, at least 90%, atleast 95%, at least 98%) a rabbit heavy or light chain-encoding nucleicacid. The percentage identity is based on the shorter of the sequencescompared. Well known programs such as BLASTN (2.0.8) (Altschul et al.(1997) Nucl. Acids. Res. 25:3389-3402) using default parameters and nofilter may be employed to make a sequence comparison.

The rabbit monoclonal antibody may be a full-length natural antibody orany chimera thereof, for example. Methods for producing chimericantibodies are known in the art. See e.g., Morrison et al (Science 1985229:1202); Oi et al (BioTechniques 1986 4:214); Gillies et al. (J.Immunol. Methods 1989 125:191-202) and U.S. Pat. Nos. 5,807,715,4,816,567 and 4,816397, which are incorporated herein by reference intheir entirety.

The amino acid sequences of the CDRs and framework regions of heavy andlight chains of 44 exemplary rabbit monoclonal antibodies thatneutralize TNFα activity are set forth in FIGS. 1A and 2A, respectively.

Modified Antibodies

The above-described rabbit monoclonal antibodies may be modified toprovide modified antibodies that neutralize TNFα activity. The modifiedantibodies may be made by substituting, adding, or deleting at least oneamino acid of an above-described rabbit monoclonal antibody. In oneembodiment, an above-described TNFα-neutralizing antibody is modified toprovide a humanized antibody for human therapeutic use, or another typeof modified antibody. In general, these modified antibodies have thegeneral characteristics of the above-described rabbit antibodies andcontain at least the CDRs of an above-described rabbit antibody, or, incertain embodiments, CDRs that are very similar to the CDRs of anabove-described rabbit antibody.

Humanized Antibodies

In one embodiment, therefore, the invention provides humanized versionsof the above-described rabbit monoclonal antibodies. In general,humanized antibodies are made by substituting amino acids in theframework regions of a parent non-human antibody to produce a modifiedantibody that is less immunogenic in a human than the parent non-humanantibody. Antibodies can be humanized using a variety of techniquesknown in the art including, for example, CDR-grafting (EP 239,400; PCTpublication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan,Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., ProteinEngineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973(1994)), and chain shuffling (U.S. Pat. No. 5,565,332). In certainembodiments, framework substitutions are identified by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions (see, e.g.,U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988)).Additional methods for humanizing antibodies contemplated for use in thepresent invention are described in U.S. Pat. Nos. 5,750,078; 5,502,167;5,705,154; 5,770,403; 5,698,417; 5,693,493; 5,558,864; 4,935,496; and4,816,567, and PCT publications WO 98/45331 and WO 98/45332. Inparticular embodiments, a subject rabbit antibody may be humanizedaccording to the methods set forth in published U.S. patent applications20040086979 and 20050033031. Accordingly, the rabbit antibodiesdescribed above may be humanized using methods that are well known inthe art.

In one embodiment of particular interest, a subject rabbit monoclonalantibody may be humanized in accordance with the methods set forth ingreat detail in U.S. patent application Ser. No. 10/984,473, filed onNov. 8, 2004 and entitled “Methods for antibody engineering”, whichapplication is incorporated by reference in its entirety. In general,this humanization method involves identifying a substitutable positionof an antibody by comparing sequences of antibodies that bind to thesame antigen, and replacing the amino acid at that position with adifferent amino acid that is present at the same position of a similarhuman antibody. In these methods, the amino acid sequence of a parentalrabbit antibody is compared to (i.e., aligned with) the amino acidsequences of other related rabbit antibodies to identify variationtolerant positions. The amino acid sequence of the variable domain ofthe parental rabbit antibody is usually compared to a database of humanantibody sequences, and a human antibody that has an amino acid sequencethat is similar to that of the parental antibody is selected. The aminoacid sequences of the parental antibody and the human antibody arecompared (e.g., aligned), and amino acids at one or more of thevariation tolerant positions of the parental antibody are substituted bycorrespondingly positioned amino acids in the human antibody.

The above-discussed variation tolerant position substitution methods arereadily incorporated into any known humanization method and are alsoreadily employed to produce humanized antibodies containing CDR regionsthat are altered with respect to the CDR regions of the parent antibody.Accordingly humanized TNFα-neutralizing antibodies containing alteredversions of the CDRs of the above-described rabbit monoclonal antibodiesare provided.

The humanized TNFα-neutralizing antibodies of the invention thereforemay contain the unaltered CDRs of an above-described rabbitTNFα-neutralizing antibody, or, in certain embodiments, altered CDRs ofan above-described rabbit TNFα-neutralizing antibody. A humanizedantibody containing altered CDRs of an above-described rabbitTNFα-neutralizing antibody generally contains CDRs having 1, up to 2, upto 3, up to 4, up to 5, up to 6, up to 7 and in certain cases up toabout 10 amino acid substitutions, as compared to the CDRs of anabove-described rabbit TNFα-neutralizing antibody. The particularsubstitutable positions of a CDR, as well as the donor amino acid thatcan be substituted into those positions, are readily apparent from U.S.Ser. No. 10/984,473, filed on Nov. 8, 2004, and the sequence alignmentsshown in FIGS. 1A and 1B.

Substitutable positions of a rabbit TNFα-neutralizing antibody, as wellthe choice of amino acids that may be substituted into those positions,are revealed by aligning the heavy and light chain amino acid sequencesof the rabbit antibodies discussed above, and determining which aminoacids occur at which positions of those antibodies. In one exemplaryembodiment, the rabbit heavy and light chain amino acid sequences ofFIGS. 1A and 1B are aligned (as shown in FIGS. 1A and 1B), and theidentity of amino acids at each position of 44 exemplary rabbitTNFα-neutralizing antibodies is determined. As illustrated in FIG. 2A(illustrating the amino acids present at each position of the heavychains of 44 exemplary rabbit TNFα-neutralizing antibodies) and 2B(illustrating the amino acids present at each position of the lightchains of 44 exemplary rabbit TNFα-neutralizing antibodies), severalsubstitutable positions, as well as the amino acids that can besubstituted into those positions, are readily identified. For example,according to this analysis, for heavy chains, Q, E or V may be employedat position 2, S or Q may be employed at position 3, and L or V may beemployed at position 4, and so on. In particular embodiments, an aminoacid at a substitutable position of a subject rabbit antibody may besubstituted by an amino acid at a corresponding position of a similarhuman antibody to humanize the rabbit antibody. In one embodiment, theamino acid of the human antibody is only substituted into the rabbitantibody if the amino acid is one of the amino acids known to be presentat that position in a different rabbit antibody that binds to the sameantigen.

In a refinement of this method and as described in greater detail inU.S. Ser. No. 10/984,473, a group of related rabbit antibodies, i.e.,antibodies that have a similar sequence and also produced by cells thathave a common B cell ancestor, are identified. The antibodies withineach group of related antibodies generally share a common ancestorantibody, and have evolved from that ancestor antibody via somatichypermutation, gene conversion and other cellular mutation-producingmechanisms that occur during affinity maturation and the final stages ofB-cell development. The amino acid sequences of the antibodies within agroup can be compared to identify substitutable positions. Asubstitutable position of an individual antibody is identified by virtueof the fact that the identity of the amino acid at that position variesbetween the individual antibodies of a group of related antibodies. Onceidentified, the amino acid at the substitutable position of anindividual antibody can be substituted for a different amino acidwithout significantly decreasing the affinity of the antibody. Sinceantibodies containing amino acid substitutions at these substitutablepositions were originally produced and effectively tested by the immunesystem of the initial immunized animal, substitution at those positionsshould be well tolerated by the antibody. Substitutable positions inCDRs and framework regions may be identified in these methods.

For example, the 44 exemplary rabbit antibodies discussed above arereadily classified by sequence to produce 7 groups of relatedantibodies, termed herein as Groups A-G. Sequence alignments of theheavy and light chains of the antibodies within each of the sevengroups, as well as an indication (*) of which positions of those heavyand light chains are substitutable, are illustrated in FIGS. 3A and 3B,respectively. If such a substitution is desirable, a humanizing aminoacid substitution may be made at any one or more (e.g., 1, up to 2, upto 3 or up to 4 or more) of those positions.

As noted above, the subject rabbit antibodies may be modified to providemodified antibodies. In particular embodiments, these methods includemaking one or more amino acid substitutions (e.g., one, up to two, up tothree, up to four or up to five of more, usually up to 10 or more). Anamino acid substitution may be at any position, and the amino acid atthat position may be substituted by an amino acid of any identity. Incertain embodiments, a modified antibody may have the same generalcharacteristics of the above-described rabbit antibodies. In oneembodiment, after a substitutable position has been identified using themethods of U.S. Ser. No. 10/984,473, the amino acids at that positionmay be substituted. In particular embodiments, an amino acidsubstitution may be a humanizing substitution (i.e., a substitution thatmake the amino acid sequence more similar to that of a human antibody),a directed substitution (e.g., a substitution that make the amino acidsequence of an antibody more similar to that of a related antibody inthe same group), a random substitution (e.g., a substitution with any ofthe 20 naturally-occurring amino acids) or a conservative substitution(e.g., a substitution with an amino acid having biochemical propertiessimilar to that being substituted).

Exemplary substitutable positions of the representative rabbitantibodies of FIGS. 1A and 1B are listed above. These positions may besubstituted without significant loss of antibody activity.

In certain embodiments, modified antibodies of the invention may containa heavy or light chain that is encoded by a polynucleotide thathybridizes under high stringency conditions to a rabbit heavy or lightchain-encoding nucleic acid. High stringency conditions includeincubation at 50° C. or higher in 0.1×SSC (15 mM saline/0.15 mM sodiumcitrate).

In certain embodiments, modified antibodies of the invention may containa heavy or light chain that is encoded by a polynucleotide that is atleast 80% identical to (e.g., at least 85%, at least 90%, at least 95%,at least 98%) a rabbit heavy or light chain-encoding nucleic acid. Thepercentage identity is based on the shorter of the sequences compared.Well known programs such as BLASTN (2.0.8) (Altschul et al. (1997) Nucl.Acids. Res. 25:3389-3402) using default parameters and no filter may beemployed to make a sequence comparison.

Methods of Using Antibodies to Inhibit TNFα Activity

Any of the antibodies described above, including the subject rabbitmonoclonal antibodies and humanized versions of the same, may beemployed in a method of inhibiting TNFα activity. The antibodies may beemployed in a variety of protocols described below.

In one embodiment, a modified antibody may be produced and tested forTNFα-neutralizing activity. In other words, in one embodiment the methodincludes altering at least 1 amino acid of a subject rabbit monoclonalantibody to produce a modified antibody, and testing the modifiedantibody for a TNFα neutralizing activity.

The protocols that may be employed in these methods are numerous, andinclude but are not limited to cell-free assays, e.g., binding assays toa TNFα receptor; cellular assays in which a cellular phenotype ismeasured, e.g., gene expression or cytotoxicity; and in vivo assays thatinvolve a particular animal (which, in certain embodiments may be ananimal model for a TNFα-related condition).

Such assays, including those described above, are well known in the artand are described in a variety of publications, including 20040151722,20050037008, 20040185047, 20040138427, 20030187231, 20040002589,20030199679, U.S. Pat. No. 6,090,382 and Balazovich (Blood 1996 88:690-696).

Methods for Producing Antibodies

In many embodiments, the nucleic acids encoding a subject monoclonalantibody are introduced directly into a host cell, and the cellincubated under conditions sufficient to induce expression of theencoded antibody.

Any cell suitable for expression of expression cassettes may be used asa host cell. For example, yeast, insect, plant, etc., cells. In manyembodiments, a mammalian host cell line that does not ordinarily produceantibodies is used, examples of which are as follows: monkey kidneycells (COS cells), monkey kidney CVI cells transformed by SV40 (COS-7,ATCC CRL 165 1); human embryonic kidney cells (HEK-293, Graham et al. J.Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);chinese hamster ovary-cells (CHO, Urlaub and Chasin, Proc. Natl. Acad.Sci. (USA) 77:4216, (1980); mouse sertoli cells (TM4, Mather, Biol.Reprod. 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70);african green monkey kidney cells (VERO-76, ATCC CRL-1587); humancervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Mather et al.,Annals N.Y. Acad. Sci 383:44-68 (1982)); NIH/3T3 cells (ATCC CRL-1658);and mouse L cells (ATCC CCL-1). Additional cell lines will becomeapparent to those of ordinary skill in the art. A wide variety of celllines are available from the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209.

Methods of introducing nucleic acids into cells are well known in theart. Suitable methods include electroporation, particle gun technology,calcium phosphate precipitation, direct microinjection, and the like.The choice of method is generally dependent on the type of cell beingtransformed and the circumstances under which the transformation istaking place (i.e. in vitro, ex vivo, or in vivo). A general discussionof these methods can be found in Ausubel, et al, Short Protocols inMolecular Biology, 3rd ed., Wiley & Sons, 1995. In some embodimentslipofectamine and calcium mediated gene transfer technologies are used.

After the subject nucleic acids have been introduced into a cell, thecell is typically incubated, normally at 37° C., sometimes underselection, for a period of about 1-24 hours in order to allow for theexpression of the antibody. In most embodiment, the antibody istypically secreted into the supernatant of the media in which the cellis growing in.

In mammalian host cells, a number of viral-based expression systems maybe utilized to express a subject antibody. In cases where an adenovirusis used as an expression vector, the antibody coding sequence ofinterest may be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing the antibody molecule ininfected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA81:355-359 (1984)). The efficiency of expression may be enhanced by theinclusion of appropriate transcription enhancer elements, transcriptionterminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544(1987)).

For long-term, high-yield production of recombinant antibodies, stableexpression may be used. For example, cell lines, which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with immunoglobulin expression cassettes and a selectablemarker. Following the introduction of the foreign DNA, engineered cellsmay be allowed to grow for 1-2 days in an enriched media, and then areswitched to a selective media. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into a chromosome and grow to form foci which inturn can be cloned and expanded into cell lines. Such engineered celllines may be particularly useful in screening and evaluation ofcompounds that interact directly or indirectly with the antibodymolecule.

Once an antibody molecule of the invention has been produced, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In many embodiments, antibodies are secretedfrom the cell into culture medium and harvested from the culture medium.

Formulations and Administration

The antibodies of the invention may be administered in any manner whichis medically acceptable. This may include injections, by parenteralroutes such as intravenous, intravascular, intraarterial, subcutaneous,intramuscular, intratumor, intraperitoneal, intraventricular,intraepidural, or others as well as oral, nasal, ophthalmic, rectal, ortopical. Sustained release administration is also specifically includedin the invention, by such means as depot injections or erodibleimplants. Localized delivery is particularly contemplated, by such meansas delivery via a catheter to one or more arteries, such as the renalartery or a vessel supplying a localized tumor.

The subject antibodies may be an a pharmaceutically acceptable carrier.The term “pharmaceutically acceptable carrier” means one or more organicor inorganic ingredients, natural or synthetic, with which the mutantproto-oncogene or mutant oncoprotein is combined to facilitate itsapplication. A suitable carrier includes sterile saline although otheraqueous and non-aqueous isotonic sterile solutions and sterilesuspensions known to be pharmaceutically acceptable are known to thoseof ordinary skill in the art. An “effective amount” refers to thatamount which is capable of ameliorating or delaying progression of thediseased, degenerative or damaged condition. An effective amount can bedetermined on an individual basis and will be based, in part, onconsideration of the symptoms to be treated and results sought. Aneffective amount can be determined by one of ordinary skill in the artemploying such factors and using no more than routine experimentation.

In one embodiment a subject antibody is administered to a patient byintravenous, intramuscular or subcutaneous injection. An antibody may beadministered within a dose range between about 0.1 mg/kg to about 100mg/kg; between about 1 mg/kg to 75 mg/kg; or about 10 mg/kg to 50 mg/kg.The antibody may be administered, for example, by bolus injunction or byslow infusion. Slow infusion over a period of 30 minutes to 2 hours maybe used.

Utility

The subject antibodies are useful for treating a TNF-α-mediateddisorder. In one embodiment, the invention provides a method of treatinga subject for a TNFα-related condition. The method generally involvesadministering a subject antibody a subject having a TNFα-relateddisorder in an amount effective to treat at least one symptom of theTNFα-related disorder.

The term “TNF-α-mediated disorder” refers to any disorder or diseasestate in which TNF-α plays a direct role, e.g., by excessive productionor release of TNF-α itself or by TNF-α-induced production or release ofanother agent that produces a pathological effect. As such, the subjectmethods are useful for treating any fibrotic disorder, includingobliterative bronchiolitis, interstitial lung disease, fibrotic lungdisease (e.g., idiopathic pulmonary fibrosis (IPF), pulmonary fibrosisof a known etiology, e.g. cystic fibrosis, adult respiratory distress,syndrome (ARDS), tumor stroma in lung disease, systemic sclerosis,Hermansky-Pudlak syndrome (HPS), coal worker's pneumoconiosis (CWP),asbestosis, sarcoidosis, silicosis, black lung disease, chronicpulmonary hypertension, AIDS associated pulmonary hypertension, and thelike), human kidney disease (e.g., nephrotic syndrome, Alport'ssyndrome, HIV-associated nephropathy, polycystic kidney disease, Fabry'sdisease, diabetic nephropathy, and the like), glomerular nephritis,nephritis associated with systemic lupus erythematosus, fibroticvascular disease, arterial sclerosis, atherosclerosis, varicose veins,coronary infarcts, cerebral infarcts, musculoskeletal fibrosis,post-surgical adhesions, cutis keloid formation, progressive systemicsclerosis, primary sclerosing cholangitis (PSC), renal fibrosis,scleroderma (local and systemic), diabetic retinopathy, glaucoma,Peyronie's disease, penis fibrosis, arethrostenosis after test usingcystoscope, inner accretion after surgery, myelofibrosis, idiopathicretroperitoneal fibrosis, fibrosis incident to microbial infection (e.g.viral, bacterial, fungal, parasitic, etc.), fibrosis incident toinflammatory bowel disease (including stricture formation in Crohn'sdisease and microscopic colitis), fibrosis induced by chemical orenvironmental insult (e.g., cancer chemotherapy, pesticides, radiation(e.g. cancer radiotherapy), and the like), peritoneal fibrosis, liverfibrosis, myocardial fibrosis, pulmonary fibrosis, Grave'sophthalmopathy, drug induced ergotism, cardiovascular disease, fibrosisincident to benign or malignant cancer (including desmoid tumor),Alzheimer's disease, scarring, scleroderma, glioblastoma in Li-Fraumenisyndrome, sporadic glioblastoma, myeloid leukemia, acute myelogenousleukemia, myelodysplastic syndrome, myeloproliferative syndrome,fibrosis incident to benign or malignant gynecological cancer (e.g.,ovarian cancer, Lynch syndrome, and the like), Kaposi's sarcoma,Hansen's disease, inflammatory bowel disease (including strictureformation in Crohn's disease and microscopic colitis), Crohn's disease,ulcerative colitis, multiple sclerosis, Type II diabetes, rheumatoidarthritis, asthma, chronic bronchitis, atopic dermatitis, urticaria,allergic rhinitis, allergic conjunctivitis, chronic obstructivepulmonary disease, graft rejection, graft-versus-host disease, sepsis,and the like.

Some of these disorders are described in greater detail below.

CNS disorders Evidence exists in the literature that TNF-α has effectson cells of the central nervous systems (CNS). Evidence for CNSproduction of TNF-α, involvement of TNF-α in brain injury, the role ofpolymorphonuclear leukocytes (PMNs) in brain injury, the role ofadhesion molecules in brain injury, and potential TNF-α directedtherapeutic strategies for prevention of brain injury have been reviewedin the literature. See, e.g., Babak Arvin et al. (1995) Ann. N.Y. Acad.Sciences 765: 62-71.

The prevention of brain edema by anti-TNF-α antibodies in experimentalmeningitis provides firm evidence for the involvement of TNF-α in thebreakdown of the Blood Brain Barrier. TNF-α can also trigger theinfiltration of neutrophils into the tissue with consequent induction ofsecondary mediators in local areas. See, e.g., “Cytokines and CNS,”Edit: R. M. Ransohoff and E. N. Beneviste, CRC Press, Page 193, 1996).

Closed head injury (CHI) in rats triggers the production of TNF-α in thecontused brain hemisphere, and it was shown that a decrease in TNF-αlevels or inhibition of its activity is accompanied by significantlyreduced brain damage. Shohami et al. (1996) J. Cerebral Blood FlowMetab., 16:378-384.

Multiple Sclerosis Multiple sclerosis (MS) plaques within the CNS areinfiltrated by peripheral blood mononuclear cells. In patients, TNF-α,but not lymphotoxin, is overproduced by peripheral blood mononuclearcells during MS relapse. Glabinski et al. (1995) Neurol Scand.91:276-279. TNF-α has an ability to cause cell death of oligodendrocytesin vitro. Robbins et al. (1987) J. Immunol., 139:2593. This aspect ofTNF-α activity may contribute directly to myelin damage and/or thedemyelination process observed in diseases such as multiple sclerosis(MS). TNF-α has been shown to play a central role in the demyelinationof the CNS in MS. Serum levels of TNF-α are elevated in patients withactive MS, and TNF-α producing macrophages, microglia and astrocytes arepresent at active lesion sites. In in vitro experiments, TNF-α directlymediates oligodendrocyte damage and suppresses myelin formation, and itstimulates astrocytes, which are then responsible for the CNS scarringplaques in MS (Owens and Sriram, Neurological Clinics, 13:51, 1995).

Serum levels of TNFα are elevated in patients with active MS (M.Chofflon et al., Eur. Cytokine Net., 3:523, 1991; Sharief, M. K. andHentgen, N. E. Jour. Med., 325:467, 1991). TNF-α producingmacrophages/microglia and astrocytes are present at active lesion sites(K. Selmaj al., Jour. Clin. Invest., 87:949, 1991). In in vitroexperiments, TNF-α directly mediates oligodendrocyte damage andsuppresses myelin formation (K. Selmaj et al., J. Immunol., 147:1522,1990); T. Tsumamoto et al., Acta Neurol. Scand., 91:71, 1995), and itstimulates astrocytes, which are responsible for the scarring plaques(K. Selmaj et al., J. Immunol., 144:129, 1990).

An increase in TNF-α expression preceding MS exacerbation attacks hasbeen shown. (“Cytokines and the CNS,” Edit: R. M. Ransohoff and E. N.Beneviste, CRC Press, 1996, p. 232). In vivo studies of murine, rat andhuman demyelinating diseases indicate that TNF-α participates in theinflammatory reactions that take place within the CNS. TNF-α positiveastrocytes and macrophages have been identified in the brains of MSpatients, particularly in the plaque region (F. M. Hofman et al., J.Exp. Med., 170:607, 1991, and Selmaj et al., J. Clin. Invest., 87:949,1991) have determined that both TNF-α and TNF-β are present in MS plaqueregions, and that TNF-α is localized within astroyctes, whereas TNF-α isassociated with microglia and T-cells. Increased serum and cerebrospinalfluid levels of TNF-α have been documented in patients with MS (Sharief,M. K., M. Phil, and R. Hentges, N. Engl. J. Med., 325:467, 1991), and astrong correlation exists between cerebrospinal fluid levels of TNF-α,disruption of the blood brain barrier, and high levels of circulatingICAM-1 in patients with active MS.

Alzheimer's Disease Alzheimer's disease (AD), the most common dementingdisorder of late life, is a major cause of disability and death in theelderly. The disease is manifested by the appearance of abnormalities inthe brain, particularly involving the hippocampus, amygdala, thalamusand neocortex. Lesions in these regions are associated withdysfunction/death of neurons and deafferentation of targets. Theprincipal pathological hallmarks of AD are deposits of the amyloid-βprotein (Aβ) in extracellular parenchyma and cerebral vessels, andneurofibrillary tangles.

TNF-α has been generally elevated in the serum of AD patients based uponboth antibody assays and bioassays. In one study almost half of the ADcases had elevated TNF-α, but none of the controls had a similarelevation. The blood-brain barrier does not normally permit passage ofcytokines. However, there is evidence to suggest that the blood-brainbarrier may not be intact in AD.

Respiratory disorders TNF-α has been shown to play a role in pulmonaryfibrosis induced by bleomycin and silica (Piguet et al., Jour. Exper.Med., 170:655-663, 1989, and Nature, 344:245-247, 1990; Everson andChandler, Amer. Jour. Path., 140:503-512, 1992; Phan and Kunkel, Exp.Lung Res. 18:29-43, 1992; also, Warren et al., Jour. Clin. Invest.,84:1873-1882, 1989; Denis et al., Amer. Jour. Cell Mol. Biol.,5:477-483, 1991). TNF-α has been reported to orchestrate itsproinflammatory effects by regulating the compartmentalized release ofsecondary messenger cytokines. Investigations have shown that nude miceexposed to chronic in vivo TNF-α develop pulmonary inflammation andfibrosis (ARRD 145:A307, 1992).

Asthma It has been reported that levels of TNF-α are increased inbronchoalveolar lavage (BAL) fluid from patients with allergic asthma.Cirelli, et al. (1995) Amer. Jour. Resp. Critical Care Med., 151:345A;Redington et al., (1995) Amer. Jour. Respir. Crit. Care Med., 151: 702A.These findings indicate an increased tissue level of TNF-α in asthma andthat this may contribute to the pathophysiology of the condition.

Chronic Obstructive Pulmonary Disease (COPD) Another disease state inwhich TNF-α plays a role in the pathophysiology is chronic obstructivepulmonary disease. In silicosis, a disease of progressive respiratoryfailure caused by a fibrotic reaction, antibody to TNF-α completelyblocked the silica-induced lung fibrosis in mice (Piguet et al., Nature,344:245-247, 1990). High levels of TNF-production (in the serum and inisolated macrophages) have been demonstrated in animal models of silicaand asbestos induced fibrosis (Bissonnette et al., Inflammation,13:329-339, 1989).

Adult Respiratory Distress Syndrome (ARDS) Excessive TNF-alpha.concentrations in excess of 12,000 pg/ml have been detected in pulmonaryaspirates from ARDS patients (Millar et al., Lancet, 2(8665):712-714,1989). Systemic infusion of recombinant TNF-α was shown to result inchanges typically seen in ARDS (Ferrai-Baliviera et al., Arch. Surg.,124:1400-1405, 1989).

Lung Sarcoidosis Alveolar macrophages from pulmonary sarcoidosispatients have been found to spontaneously release massive quantities ofTNF-α as compared with macrophages from normal donors (Baughman et al.,Jour. Lab. Clin. Med., 115:36-42, 1990). TNF-α also implicated in otheracute disease states such as the pathophysiologic responses whichfollows subsequent reperfusion. It is involved in reperfusion injury,and is a major cause of tissue damage after loss of blood flow. (Vedderet al., Proc. Nat. Acad. Sci., 87:2643-2646, 1990).

Sepsis Overproduction of TNF-α has been implicated in the pathogenesisof endotoxin induced septic shock, (see Carswell et al., Proc. Nat.Acad. Sci., 2:3666-3670, 1975). Endotoxin is the lipopolysaccharidecomponent of the cell wall of gram-negative bacteria, and is amacrophage activator which induces the synthesis and enhanced secretionof TNF-α and other biologically active cytokine molecules. TNF-α isrecognized as a central mediator of sepsis, septic shock and multipleorgan failure. These host reactions are associated with increased bloodlevels of TNF-α, due to increased TNF-α production. (F. Stuber et al.,Jour. Inflam., 46:42-50, 1996).

Liver disorders Because of its central role in metabolism and hostdefense mechanisms, the liver is thought to be major organ responsiblefor initiation of the multiple organ failure during sepsis. Thedepression in hepatocellular function in early, hyperdynamic stages ofsepsis does not appear to be due to any reduction in hepatic perfusion,but is associated with elevated levels of circulating cytokines such asTNF-α. Furthermore, administration of recombinant TNF-α at doses that donot reduce cardiac output or hepatic perfusion, produces hepatocellulardysfunction. (P. Wang et al., Amer. Jour. Physiol., 270:5, 1996).

The role of TNF-α in induction of hepatic apoptosis undertranscriptional arrest, activation of the 55 kDa receptor in theinduction of hepatic apoptosis, the glycosylation step in TNF-inducedhepatic apoptosis, hepatic injury induction by T cell-initiated cytokinerelease, and Ta cell-dependent TNF-mediated liver injury withouttranscriptional arrest has been reported. (A. Wendel et al., Cell. Biol.Mol. Basis. Liver Transp., Int., Ringberg Conf. Hepatic Transp., 2nd,1995, Pages 105-111).

Diabetes TNF-α plays a central role in the state of insulin resistanceassociated with obesity. It has been previously shown that one importantmechanism by which TNF-α interferes with insulin signaling is throughserine phosphorylation of insulin receptor substrate-1 (IRS-1), whichcan function as an inhibitor of the tyrosine kinase activity of theinsulin receptor (IR). The data strongly suggest that TNF-α inhibitssignaling via a stimulation of p55 TNFR, and sphingomyelinase activity,which results in the production of an inhibitory form of IRS-1 (Peraldiet al., J. Biol. Chem. 271:13018-13022, 1996).

Crohn's disease TNF-α levels are elevated in Crohn's disease. In onestudy, TNF-α concentrations were measured in stool samples from normalchildren, infants with diarrhea, and children with inflammatory boweldisease in active and inactive phases. Compared with diarrhea controls,stool TNF-α concentrations were significantly increased in children withactive Crohn's disease. In patients with inactive Crohn's disease,either as a result of surgery, or treatment with steroids, theconcentration of stool TNF-α fell to the level of the controls (C. P.Braegger et al., Lancet, 339:89-91, 1992).

Pre-Eclampsia Pre-eclampsia is an endothelial disorder, and TNF-α hasfundamental effects on endothelial cells by several means, includingalteration of the balance between oxidant and anti-oxidant, changing thepattern of prostaglandin production, and affecting the expression ofseveral cell surface components. In patients, results show that TNF-αmRNA expression is significantly elevated in preeclamptic patientscompared to the control groups. These observations are consistent with amajor role for TNF-α in the development of eclampsia (G. Chen et al.,Clin. Exp. Immunol. 104:154-159, 1996).

Dermal Burns The protein catabolic rate and TNF-α content of the soleusmuscle of the scalded region and remote region were dynamicallydetermined in the first week after the rats were inflicted with 37% TBSAfull thickness scalding. The TNF-α content of skeletal muscles was fargreater in the scalded region than in the remote region. TNF-.alpha.increase was also significantly correlated to the protein catabolic rateof the skeletal muscles (Li et al., Jour. Med. Coll., PLA 10:262-267,1995; C.A. 125:938, 1245:8156a, 1996).

Bone Resorption TNF-α is increased in bone resorption diseases,including arthritis, wherein it has been determined that when activated,leukocytes will produce a bone reabsorbing activity. Data indicate thatTNF-α enhances this activity (Bertolini et al., Nature, 319:516-518,1986, and Johnson et al., Endocrinology, 124:1424-1427, 1989). TNF-αstimulates bone resorption and inhibits bone formation in vitro and invivo through stimulation of osteoclast formation and activation combinedwith inhibition of osteoblast function. TNF-α may be involved in manybone resorption diseases, including arthritis.

Rheumatoid Arthritis Analysis of cytokine mRNA and protein in humanrheumatoid arthritis tissue revealed that many proinflammatory cytokinessuch as TNF-α are abundant in all patients regardless of therapy. Inrheumatoid joint cell cultures that spontaneously produce IL1, TNF-α wasthe major dominant regulator of IL1. Subsequently, other proinflammatorycytokines were also inhibited if TNF-α was neutralized, leading to theconcept that the proinflammatory cytokines were linked in a network withTNF-α at its apex. This led to the concept that TNF-α was of majorimportance in rheumatoid arthritis. This has been successfully tested inanimal models of collagen-induced arthritis, and these studies haveprovided the rationale for clinical trials of anti-TNF-α therapy inpatients with long-standing rheumatoid arthritis. Several clinicaltrials using a chimeric anti-TNF-α antibody have shown marked clinicalbenefit, verifying the concept that TNF-α is of major importance inrheumatoid arthritis. Re-treatment clinical studies have also shownbenefit in repeated relapses, indicating that the disease remains TNF-αdependent (M. Feldmann, Annual Rev. Immunol., 14:397-440, 1996).

Vascular disorders TNF-α alters the properties of endothelial cells andhas various pro-coagulant activities, such as production of an increasein tissue factor procoagulant activity and suppression of theanticoagulant protein C pathway as well as down-regulating theexpression of thrombomodulin (Sherry et al., Jour. Cell. Biol.,107:1269-1277, 1988). TNF-α has activities which, together with itsearly production (during the initial stages of a trauma or injuryevent), make it a mediator of response to tissue injury in severalimportant disorders including, but not limited to myocardial infarction,stroke and circulatory shock. Of specific importance may be TNF-αinduced expression of adhesion molecules, such as intercellular adhesionmolecule (ICAM) or endothelial leukocyte adhesion molecule onendothelial cells (Munro et al., Am. Jour. Path., 135:121-132, 1989).

Cardiac disorders Evidence indicates that the current top suspects inheart failure are noradrenaline, angiotensin, vasopressin, endothelin,and tumor-necrosis factor (TNF-.alpha.), (N.E. J. Med., 323:236-241,1990). It has been reported that concentrations of TNF-α, which causecachexia in chronic inflammatory disorders, infections, cancer and otherdiseases, are elevated in patients with severe heart failure, especiallythose with the more severe manifestations of the disease, such ascardiac cachexia.

Graft vs. host disease In graft versus host reactions, increased serumTNF-α levels have been associated with major complications followingacute allogenic bone marrow transplants (Holler et al., Blood,75:1011-1016, 1990).

An subject antibody modulates, i.e., reduces or increases a symptom ofthe animal model disease or condition by at least about 10%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 55%, at least about 60%, at least about 65%, at least about 70%,at least about 80%, at least about 90%, or more, when compared to acontrol in the absence of the antibody. In general, a subjet antibodywill cause a subject animal to be more similar to an equivalent animalthat is not suffering from the disease or condition. Monoclonalantibodies that have therapeutic value that have been identified usingthe methods and compositions of the invention are termed “therapeutic”antibodies.

Kits

Also provided by the subject invention are kits for practicing thesubject methods, as described above. The subject kits at least includeone or more of: a subject antibody, a nucleic acid encoding the same, ora cell containing the same. The subject antibody may be humanized. Otheroptional components of the kit include: buffers, etc., for administeringthe antibody or for performing a TNFα activity assay. The nucleic acidsof the kit may also have restrictions sites, multiple cloning sites,primer sites, etc to facilitate their ligation to non-rabbit antibodynucleic acids. The various components of the kit may be present inseparate containers or certain compatible components may be precombinedinto a single container, as desired.

In addition to above-mentioned components, the subject kits typicallyfurther include instructions for using the components of the kit topractice the subject methods. The instructions for practicing thesubject methods are generally recorded on a suitable recording medium.For example, the instructions may be printed on a substrate, such aspaper or plastic, etc. As such, the instructions may be present in thekits as a package insert, in the labeling of the container of the kit orcomponents thereof (i.e., associated with the packaging or subpackaging)etc. In other embodiments, the instructions are present as an electronicstorage data file present on a suitable computer readable storagemedium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actualinstructions are not present in the kit, but means for obtaining theinstructions from a remote source, e.g. via the internet, are provided.An example of this embodiment is a kit that includes a web address wherethe instructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

Also provided by the subject invention is are kits including at least acomputer readable medium including programming as discussed above andinstructions. The instructions may include installation or setupdirections. The instructions may include directions for use of theinvention with options or combinations of options as described above. Incertain embodiments, the instructions include both types of information.

Providing the software and instructions as a kit may serve a number ofpurposes. The combination may be packaged and purchased as a means forproducing rabbit antibodies that are less immunogenic in a non-rabbithost than a parent antibody, or nucleotide sequences them.

The instructions are generally recorded on a suitable recording medium.For example, the instructions may be printed on a substrate, such aspaper or plastic, etc. As such, the instructions may be present in thekits as a package insert, in the labeling of the container of the kit orcomponents thereof (i.e., associated with the packaging orsubpackaging), etc. In other embodiments, the instructions are presentas an electronic storage data file present on a suitable computerreadable storage medium, e.g., CD-ROM, diskette, etc, including the samemedium on which the program is presented.

It is evident from the above discussion that the subject inventionprovides important new TNFα-neutralizing antibodies. Accordingly, thepresent invention represents a significant contribution to the art.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A monoclonal antibody comprising: a) a variable domain comprising: i.a heavy chain variable region comprising a CDR1 region identical toamino acid residues 30-34 of SEQ ID NO: 31, a CDR2 region identical toamino acid residues 49-64 of SEQ ID NO: 31 and a CDR3 region identicalto amino acid residues 97-109 of SEQ ID NO: 31; and ii. a light chainvariable region comprising a CDR1 region identical to amino acidresidues 23-33 of SEQ ID NO: 75, a CDR2 region identical to amino acidresidues 49-55 of SEQ ID NO: 75 and a CDR3 region identical to aminoacid residues 88-99 of SEQ ID NO: 75; or b) a variant of said variabledomain of part a) that is otherwise identical to said variable domain ofpart a) except for up to 10 amino acid substitutions in said CDRregions; wherein said monoclonal antibody neutralizes TNFα activity. 2.The monoclonal antibody of claim 1, wherein said monoclonal antibodycomprises: a variant of said variable domain of part a) that isotherwise identical to said variable domain of part a) except for 1 to 7amino acid substitutions in said CDR regions.
 3. The monoclonal antibodyof claim 1, wherein said monoclonal antibody comprises: a variant ofsaid variable domain of part a) that is otherwise identical to saidvariable domain of part a) except for 1 to 5 amino acid substitutions insaid CDR regions.
 4. The monoclonal antibody of claim 1, wherein theamino acid sequences of the framework regions of the heavy and lightchain variable regions are different from the framework regions of SEQID NO: 31 and SEQ ID NO: 75, respectively.
 5. The monoclonal antibody ofclaim 4, wherein the amino acid sequences of the framework regions ofthe heavy and light chain variable regions are different from theframework regions of SEQ ID NO: 31 and SEQ ID NO: 75, respectively, dueto humanization of said monoclonal antibody.
 6. The monoclonal antibodyof claim 1, wherein said monoclonal antibody is a humanized antibody. 7.The monoclonal antibody of claim 1, wherein said monoclonal antibody isa monovalent antibody.
 8. The monoclonal antibody of claim 1, whereinsaid monoclonal antibody is a bivalent antibody.
 9. The monoclonalantibody of claim 1, wherein said monoclonal antibody is a single chainantibody.
 10. The monoclonal antibody of claim 1, wherein saidmonoclonal antibody is a Fab, Fv, scFv, or Fd fragment.
 11. Themonoclonal antibody of claim 1, wherein said monoclonal antibody is achimeric antibody.
 12. The monoclonal antibody of claim 1, wherein saidmonoclonal antibody is a fusion protein.
 13. The monoclonal antibody ofclaim 1, wherein said monoclonal antibody is detectably labeled.
 14. Themonoclonal antibody of claim 1, wherein the monoclonal antibody isconjugated to another moiety.
 15. The monoclonal antibody of claim 1,wherein said monoclonal antibody comprises a human constant domain. 16.The monoclonal antibody of claim 1, wherein said monoclonal antibodyneutralizes TNFα activity with an IC₅₀ of 1×10⁻⁷ M or less.
 17. Themonoclonal antibody of claim 1, wherein said monoclonal antibodyneutralizes TNFα activity with an IC₅₀ of 1×10^(˜)9 M or less.
 18. Themonoclonal antibody of claim 1, wherein said monoclonal antibody bindsTNFα with a K_(D) of 10⁻⁷ M or less.
 19. A composition comprising: a)the monoclonal antibody of claim 1; and b) a pharmaceutically-acceptableexcipient.